Method and device for collecting and preserving cells for analysis

ABSTRACT

The claimed subject matter comprises a device to collect and preserve cells comprising of: (1) a collection container comprised of a tube having an open end and a closed end, a closure in the open end of the tube, a vacuum drawn to a predetermined level inside the container; and (2) compounds including an anticoagulant agent and a fixative agent, wherein the compounds are in a sufficient amount to preserve said cells” original morphology and antigenic sites without significant dilution of said cells, and thereby allowing said cells to be directly analyzed by a flow cytometer without further treatment. The claimed subject matter further comprises of a method of making a collection device for cells comprising of: (1) providing a tube having an open end and a closed end; (2) preloading compounds including: an anticoagulant agent, and a fixative agent into the tube, wherein the compounds are in a sufficient amount to preserve the cells” original morphology and antigenic sites without significant dilution of the cells, and thereby allowing the cells to be directly analyzed by a flow cytometer without further treatment; (3) inserting a closure into the open end of the tube; and (4) drawing a vacuum inside the tube to a predetermined level to form the collection device.

BACKGROUND

In biological and biochemical analysis, and related arts, it is oftennecessary to collect and preserve biological tissues (i.e., cells andcellular components), for useful periods of time. The collected andpreserved cells are often utilized in a wide variety of applications,including but not limited to instructional aids and the diagnosis andtreatment of diseases. For example, such cells are often utilized inhistological, cytological, immunological, and proteinaceous studies andthe like.

Various methods are known in the art for analyzing histological,cytological, immunological, and proteinaceous materials. For example,surface marker analysis has developed as a laboratory tool, which isparticularly useful for clinical diagnosis through the investigation ofimmunostates, differentiation of cell types and development stages, andother cell processes. The expansion of uses for surface marker analysishas resulted in the use of flow cytometry and antibody probes toevaluate cellular properties. While other means of assaying for surfacemarker analysis exist, flow cytometry provides rapid, objective andquantitative assessment of surface markers. Furthermore, even though themicroscope is still the conventional means for examining preserved andstained biological materials, biological materials may also be examinedwith a flow cytometer. The flow cytometer is an important method forexamining a plurality of cells in a brief time.

Flow cytometry and flow cytometers are generally described in Keran'stext, Flow cytometry in Clinical Diagnosis (1989). Flow cytometersoperate in principle by multi-parameter analysis of heterogeneous cellpopulations (or cellular components) on a cell-by-cell basis. Flowcytometry allows biological and physical properties of cells andcellular components to be determined. In flow cytometry, cells insuspension are forced single file, via hydrodynamic focusing, throughthe path of a laser beam. Interaction of the cells with the laser beamscatters some of the light and causes excitation and emission fromfluorescent molecules present on the surface or interior of the cell. Aseries of lenses collect the scattered or emitted light and pass it to aphotomultiplier. Each photomultiplier measures a separate parameter.Parameters measured include: forward light scatter, which measuresrelative particle size; side light scatter, which measures relativegranularity or other internal structure; and fluorescence emission. Theoptical signals are converted by a computer to a data display foranalysis and interpretation. Cells collected and preserved usingconventional methods and instruments generally require further dilutionand/or treatment before they can be analyzed by flow cytometry. Thus, itis desirable in the art to obtain a method and a collection device thatallow the cells to be directly analyzed by flow cytometry withoutfurther dilution and/or treatment. (There is need for a method tocollect and transport human blood specimens for flow cytometricanalysis. Current methods are inadequate in that the samples have to beanalyzed soon after collection.)

The primary objective of tissue preservation is to provide as muchstructural detail of cells and components thereof as possible. To dothis, it is necessary to maintain the cells in their original unalteredmorphology so that maximum cellular detail may be observed. With theclinical application of immunostaining, there is also the requirementthat antigens are not altered by the method of preservation. Thus, it isdesirable in the art to obtain a method and a collection device thatmaintain the cells in their original unaltered morphology and preservetheir anti-genic sites.

The usual formulations for preservation of cells contain one or moreagents, which react vigorously with the proteins of the cells todenature and insolubilize the components of the cell. Typical of thistype of agent is picric acid, mercuric ions, formaldehyde andglutaraldehyde. In addition, some less toxic compounds can also beutilized which denature and stabilize the proteins such as acetic andformic acid. Unfortunately, the toxicity associated with such compoundsrenders their use less than satisfactory. For example, a 37% solution offormaldehyde, the most common of these fixatives, is a noxious gas whichis also toxic, flammable, and carcinogenic. Although efforts are madewhen this chemical is used to protect workers and avoid contamination ofthe drainage system when disposed, these efforts are usually bothexpensive and inconvenient, and fixatives such as formaldehyde stillpresent a danger to laboratory workers and health care professionals.Thus, it is highly desirable to develop a method and a collectiondevice, which can preserve the cells in a low toxicity and non-flammableenvironment so that it can be used safely, effectively and convenientlyin histological and other studies.

For even easier handling, it is also desirable to develop a method and acollection device that allow transportation (e.g., from the collectionsite to the analysis site) of the cells in ambient temperature.

SUMMARY

The claimed subject matter addresses many of the challenges encounteredwhen using conventional methods and instruments to collect and preservecells by providing a method and a collection device that are capable ofmaintaining the cells in their original unaltered morphology; preservingthe cell antigenic sites; and allowing the cells to be transported atambient temperature, to be handled in a low toxicity and non-flammableenvironment, and to be directly analyzed by flow cytometry withoutfurther dilution and/or treatment. The claimed subject matter morespecifically relates to a method and a device that allow cells (e.g.,whole blood, epithelial cells, spinal fluid, and the like.) to becollected and preserved for analysis and addresses many of thechallenges encountered when using conventional methods and instruments.Specifically, the claimed subject matter describes a method and acollection device that (1) use a less toxic and non-flammable reagentfor fixing and stabilizing cells; (2) allow the cells to stay in theiroriginal unaltered morphology; (3) allow the cell antigenic sites to bepreserved for a useful period of time; (4) allow the cells to betransported at ambient temperature; and/or (5) allow the cells to bedirectly analyzed by flow cytometry without further dilution and/ortreatment.

The claimed subject matter includes a device to collect and preservecells comprising of: (1) a collection container comprised of a tubehaving an open end and a closed end, a closure in the open end of thetube, a vacuum drawn to a predetermined level inside the container; and(2) compounds including an anticoagulant agent and a fixative agentselected from the group consisting of: diazolidinyl urea, imidazolidinylurea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea,2-bromo-2.-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo [3.3.0]octane,5-hydroxymethyl-1-1aza-3,7dioxabicyclo [3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7dioxabic yclo [3.3.0]octane,quaternary adamantine and combinations thereof. The claimed subjectmatter may optionally include polyacrylic acid or a suitable acid havinga pH ranging from about one to about seven inside the tube.

The compounds of the device must be in a sufficient amount to preservethe collected cells' original morphology and antigenic sites withoutsignificant dilution of the cells (i.e., in a volume that is notclinically significant), and thereby allowing the cells, stored with thecompounds, to be directly analyzed by a flow cytometer.

The claimed subject matter also includes a method comprised of (1)providing a tube with an open end and a closed end, (2) preloading thetube with compounds including: an anticoagulant agent, a fixative agentselected from the group consisting of: diazolidinyl urea, imidazolidinylurea, dimethoylol-5,5-dimethylhydantoin, dimethylol urea,2-bromo-2-nitropropane-1,3-diol, oxazolidines, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane,5-hydroxymethyl-1-1aza-3,7-dioxabicyclo[3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7dioxabic yclo[3.3.0]octane,and quaternary adamantine, and optionally a polyacrylic acid or asuitable acid having a pH ranging from about one to about seven, whereinthe compounds are in a sufficient amount to preserve the collectedcells' original morphology and antigenic sites without significantdilution of the cells, and thereby allowing the cells, stored with thecompounds, to be directly analyzed by a flow cytometer; inserting aclosure into the open end of the tube; and drawing a vacuum to apredetermined level inside the tube.

The method and device of the claimed subject matter may also optionallyinclude other art-disclosed components conventionally used in cellcollection and analysis such as gauze, glove, tourniquet, lancet,needle, test strip (e.g., immunoassay), alcohol swab, tube holder,additional cell collection tubes (with or without conventional cellanalysis additives inside these tubes), adhesive strip, syringe, glassor plastic strip, packaging means to store the desired components andthe device, and packaging means to transport at least the collected andpreserved cells stored in the device. The method of the claimed subjectmatter may also optionally include additional art disclosed methods andinstruments used for cell analysis such as a flow cytometer, ahematology analyzer, and other hematology instruments, etc.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A cross-sectional illustration of an exemplary embodiment of thecollection device of the claimed subject matter; and

FIG. 2 A flow diagram illustrating a method for making the collectiondevice illustrated in the FIG. 1.

DETAILED DESCRIPTION

The claimed subject matter can be satisfied by embodiments in manydifferent forms, the drawings and the description herein describe indetail a preferred embodiment of the claimed subject matter. It isunderstood that the present disclosure is to be considered exemplary ofthe principles of the claimed subject matter and is not intended tolimit the claimed subject matter to the embodiment illustrated. Thescope of the claimed subject matter is measured by the appended claimsand their equivalents.

Turning now to the drawings, FIG. 1 shows a cross-sectional illustrationof a device 100 that incorporates a preferred embodiment of this claimedsubject matter and can be used to collect and preserve biologicaltissues such as cells and cellular components for analysis. The device100 is particularly useful in the collection of whole blood, but can beuse to collect other types of bodily fluids and/or biological tissues(e.g., epithelial cells, bone marrow, spinal fluid and the like)including, without limitation, abnormal tissue samples such asleukemias, cancer tissue cancer, and the like as long as the tissuesamples can be transformed into a cellular suspension.

The device 100 includes an evacuated collection container comprised of(1) a tube 12 having an open end 14 and a closed end 16; a closure(e.g., stopper) 18 in the open end of the tube 12, and a predeterminedlevel of vacuum (not shown) inside the container 10. It is preferredthat the tube 12 is made of a transparent material such as glass orplastic for better visibility. It is also preferred that the tube 12 hasan interior surface that is sterile and resists adherence to the cells20 (not shown) during collection, storage, and analysis. The closure 18is preferably puncturable by a needle and resealable allowing easytransfer of the cells 20 (e.g., the cells 20 from its host to thecontainer 10 and from the container 10 to another substrate if desired).It is also preferred that the closure 18 and the tube 12 together form aseal capable of maintaining a pressure differential between atmosphericpressure and a pressure less than atmospheric pressure within the tube18.

The size of the container 10 is not narrowly critical and is dependentupon the cell sample volume that is desired to be collected andpreserved. For example, a typical size for the container 10 may have aninternal volume of between 100 μl to 10 ml. The container 10 can beconstructed using art-disclosed methods and is commercially available(e.g., Vacutainer Plus Plastic Tubes with Hemogard Closure availablefrom Becton Dickinson and Company located in Franklin Lakes, N.J.; theevacuated sample collection tube described in U.S. Pat. No. 5,860,937,which is incorporated by reference). Of course, it should be understoodthat a wide range of changes and modifications can be made to thepreferred embodiment described above for the container 10.

For preservation (e.g., fixation, stabilization and the like) of thecells 20, the device 100 further includes compounds 22 including ananticoagulant agent 24, a fixative agent 26 selected from the groupconsisting of: diazolidinyl urea, imidazolidinyl urea,dimethoylol-5,5-dimethylhydantoin, dimethylol urea,2-bromo-2-nitropropane-1,3-diol, oxaizolidines, sodium hydroxymethylglycinate, 5-hydroxymethoxymethyl-1-1aza-3, 7dioxabicyclo [3.3.0]octane,5-hydroxymethyl-1-1aza-3,7-dioxabicyclo [3.3.0]octane,5-hydroxypoly[methyleneoxy]methyl-1-1aza-3,7-dioxabi cyclo[3.3.0]octane, and quaternary adamantine, and optionally a polyacrylicacid 28 or any suitable acid having a pH ranging from about one to aboutseven, wherein the compounds are in a sufficient amount to preserve thecollected cells' original morphology and antigenic sites withoutsignificant dilution of the cells 20, and thereby allowing the cells 20,stored with the compounds 22, to be directly analyzed by a flowcytometer. It is preferred that the compounds 22 have been sterilized(e.g., by sterilizing filtration).

A suitable amount of any art-disclosed anticoagulant agent such asethylene diamine tetra acetic acid (EDTA) and its salts, ethylene glycoltetra acetic acid (EGTA) and its salts, hirudin, heparin, citric acid,salts of citric acid, oxalic acid, salts of oxalic acid, or mixturesthereof may be used. A preferred anticoagulant agent 24 is K₃EDTA. Thesuitable amount of the anticoagulant agent 24 for the claimed subjectmatter is that effective to prevent coagulation of the cells 20 (e.g.,whole blood) without causing significant dilution of the cells 20 (i.e.,not clinically significant), and thereby allowing the cells 20, storedwith the compounds 22, to be directly analyzed by a flow cytometer). Forexample, in a preferred embodiment, K₃EDTA is the anticoagulant agent 24and its concentration weight/volume is preferably less than about 0.3g/ml, more preferably less than about 0.2 g/ml, and most preferablyabout less than about 0.15 g/ml.

The preferred fixative agent 26 is a heterocyclic urea (e.g.,diazolidinyl urea (known as DU), imidazolidinyl urea (known as IDU) or amixture thereof). The most preferred fixative agent is diazolidinylurea. The suitable amount of the fixative agent 26 for the claimedsubject matter is that effective to fix or stabilize the cells 20without causing significant dilution of the cells 20 (i.e., notclinically significant), and thereby allowing the cells 20, stored withthe compounds 22, to be directly analyzed by a flow cytometer. Forexample, in a preferred embodiment, diazolidinyl urea is the fixativeagent 26 and its concentration weight/volume is preferably about lessthan about 1 g/ml, more preferably less than about 0.75 g/ml, and mostpreferably less than about 0.5 g/ml concentration of solution of DUbefore blood sample is added.

It is known that the acid 28 may rise signal to noise ratio during flowcytometry; and therefore, the acid 28 may be optionally added as one ofthe compounds 22 in the device 100. The preferred acid 28 is apolycarboxylic acid, and more preferably a polyacrylic acid with amolecular weight of 5,000. The suitable amount of the acid 28 for theclaimed subject matter is that effective to rise signal to noise ratioduring flow cytometry but without causing significant dilution to thecells 20 (i.e., not clinically significant) so that the cells 20, storedwith the compounds 22, can be directly analyzed by a flow cytometry. Forexample, in a preferred embodiment, polyacrylic acid with a molecularweight of 5,000 is included in the container 10.

Additional compounds may optionally be added as one of the compounds 22in the device 100. Such additional and optional compounds may include:cell permeabilizing agents for substantially gaining access tointracellular analytes/epitopes and/or for lysing red blood cells;proteins that substantially protect the cells during processing and/orsubstantially reduce non-specific binding of probes; serum/lipoproteinsthat substantially protect cells during processing and/or substantiallyreduce non-specific binding of probes; RNAse inhibitors whichsubstantially inhibit digestion of RNA and/or substantially maintain RNAintegrity; nucleic acid stabilizers which substantially inhibit thedegradation of nucleic acids and nucleic acid containing compounds;amino acids/polypeptides which substantially enhance binding ofprobes/antibodies to epitopes and/or substantially increases theobservable signal; fixatives which substantially preserve cell integrityespecially for permeabilization agents, and may preserve some epitopes;anticoagulants which substantially decreases clotting of red bloodcells, chelates calcium and/or may help maintain WBCintegrity/viability; protease inhibitors which substantially decreasesdegradation of protein epitopes; antioxidants/reducing agents whichsubstantially prevent hemolysis of red blood cells and/or substantiallyprevent oxidation of peptides, and/or substantially maintain epitopes;nucleic acid dyes that generally serve to label/identify nucleic acid;carbohydrates which substantially maintain cellular integrity and/orosmolarity; and, polyacrylic acids which substantially enhance thebinding of probes and/or antibodies to epitopes; and/or substantiallyincreases signal. One of skill in the art should be able to determinethe usefulness and quantities of such optional compounds by routinetesting and knowledge of the art. Within multiple specific embodimentsthe above additional and optional compounds may be more specificallyinclude: Cell permeabilizing agents such as: DMSO (Dimethyl Sulfoxide),Ethylene glycol, Polyethylene glycol, Glycerin, Cellosolves (ethyleneglycol dimethyl ether) (phenoxyethanol), Triton X 100, Triton X 705(non-ionic detergents), 1-methyl-2-pyrrolidinone, Tween 20, Tween 40(non-ionic), Brij 35 (detergent), Polyoxyethylene ether (Polyox), Sodiumcholate, Ethylene oxide polymers, Monensin, Monactin, Pentachlorophenol,2,4 dinitrophenol, saponin, SDS (sodium dodecyl sulfate); Proteins suchas: Biotin, Albumins (egg, bovine), Gelatin, and similar such compoundsas should be known to one of skill in the art; RNAse inhibitors such as:human placenta derived RNAse inhibitor, and similar such compoundsshould be known to one of skill in the art; Nucleic acid stabilizerssuch as: Guanidinium hydrochloride, Polycations such asPolyethylenimine), and similar such compounds as should be known to oneof skill in the art; Amino acids/polypeptides such as: Glutamic acid,Glycine, Aspartic acid, and similar such compounds as should be known toone of skill in the art; Fixatives such as: Aldehydes (formaldehyde andglutaraldehyde), Alcohols (ethanol, methanol), and similar suchcompounds as should be known to one of skill in the art; Anticoagulantssuch as: EDTA (Ethylene Diamine Tetra acetic acid.), and similar suchcompounds as should be known to one of skill in the art; ACD (AcidCitrate Dextrose), Heparin, and similar such compounds as should beknown to one of skill in the art; Protease Inhibitors such as: EDTA,PMSF (phenyl methyl sulfonyl fluoride), AEBSF (2-Aminoethyl benzenesulfonyl fluoride), and similar such compounds as should be known to oneof skill in the art; Antioxidants/Reducing agents such as: Trolox,a-tocopherol, B-mercaptoethanol, and similar such compounds as should beknown to one of skill in the art; Nucleic Acid Dyes such as: DAPI(Diamidino 2-phenylindole), Propidium Iodide, Fluorescein diacetate, andsimilar such compounds as should be known to one of skill in the art;Carbohydrates such as: Sugars (sucrose), cellulose, and similar suchcompounds as should be known to one of skill in the art. It should beappreciated that the above specific listings of compounds may contain ameasure of overlap, which recognizes the sometimes-overlapping functionof certain specific compounds. One of skill in the art should understandand appreciate this aspect of the disclosure.

The claimed subject matter allows the final composition to betransported in ambient temperature. Thereafter, it is preferred that thefinal composition 30 be stored at temperature less than about 40° C. Thecells 20 stored in the final composition 30 have more than about 3 days,preferably more than about 5 days, more preferably more than about 7days stability. The claimed subject matter allows the cells 20 stored inthe final composition 30 to be directly analyzed by a flow cytometerwithout further dilution and/or treatment because the compounds 22 canpreserve the cells 20 without significantly diluting the cells 20. Anysignificant dilution of the cells 20 is likely to cause error in flowcytometry measurements (e.g., lowering the lymphocytes' count). To avoidsignificant dilution, the compounds 22 (comprising of the anticoagulantagent 24, the fixative agent 26, and optionally, the acid 28) are inconcentrated forms, preferably in a ratio with the final composition 30that is less than about 2:100, more preferably less than about 1.5:100,and most preferably less than about 1:100.

The device 100 may be included in a kit of the claimed subject mattercontaining components 32 (not shown) conventionally used to collect andanalyze the cells 30 such as alcohol swab, gauze, tube holder,tourniquet, glove, other cell collection tube (with or withoutconventional cell analysis additives inside such tube), needle (withhub, part of a syringe assembly including barrel and plunger, or withwings connected via a hub and tubing to another needle for delivery tothe device 100 or other collection tubes), lancet, adhesive strip,syringe, test strip (allowing the cells 20 to flow directly onto a glassor plastic strip containing reagents for cell analysis), glass orplastic strip containing reagents for cell analysis (e.g., immunoassay),packaging means (e.g., plastic bag, compartmentalized plastic enclosure,and the like) to store the desired components 32 and the device 100, andpackaging means to transport the cells 20 stored in the device 100 aftercollection. It is preferred that the packaging means and any othercomponents 32 that may become in physical contact with the cells 20 besterilized and the packaging means is constructed to maintain thissterile environment.

Unlike the typical histological fixing agents, the fixative agent 26 ofthe claimed subject matter has extremely low toxicity. For example,toxicity studies comparing diazolidinyl urea with formaldehyde of theprior art show the following:

Inhalation Toxicity Dermal Toxicity LD50 Formaldehyde 500 mg/kg  270mg/kg  800 mg/kg Diazolidinyl urea None 2000 mg/kg 2570 mg/kg

This reduced toxicity makes disposal and handling less of a problem. Inaddition, since there is no inhalation toxicity, there are no badgedetection devices required as there are for formaldehyde.

Another advantage offered by the fixative agent 26 is the fact that itis not flammable and therefore does not present a fire hazard as do manyof the prior art fixative agents.

The mechanism by which the fixative agent 26 provides the desired tissueand cell membrane stabilization is not known for certain. It is believedthat the fixative agent binds in some fashion to the cell membrane ortissue. This hypothesis is drawn because many members of the activeagent are known disinfectants, which kill bacteria by binding to cellstructure. This is not a full explanation of the mechanism responsiblefor the results of the claimed subject matter since many otherdisinfectants such as KATHON and OMADINE fail to provide tissue and cellstabilizing effects.

The ability of the fixative agent 26 to preserve antigenic sites is alsonot understood but it is probably due to a difference in the reactionbetween proteins and the fixative agent 26 compared to prior artpreservatives such as formaldehyde. Formaldehyde cross-links with itselfand proteins to obscure the antigen. To determine if this is true,diazolidinyl urea was added to the protein, albumin. After incubation ofthe diazolidinyl urea and protein mixture for 24 hours, disc-gelelectrophoresis indicated no change in the rate of migration of theprotein. When this experiment is conducted with formaldehyde, a largenumber of insoluble proteins result and the electrophoretic migration isaltered.

Referring to FIG. 2, a method of making the device 100 of the claimedsubject matter is comprised of providing the tube 12 having the open end14 and the closed end 16 (202). It is preferred that the tube 12 issterile. The method is further comprised of preloading (i.e.,introducing) the compounds 22 comprising of the anticoagulant agent 24,the fixative agent 26, and optionally the acid 28 into the tube 12 usingart-disclosed methods (204). The types and amounts of the anticoagulantagent 24, the fixative agent 26, and optionally, the acid 28 includingthe ratio between the compounds 22 and the final composition 30 are thesame as described above for the device 100 of the claimed subjectmatter. It is preferred the compounds have been sterilized (e.g., bysterile filtration). The method of the preloading step 204 mayoptionally include freeze drying the compounds in the tube 12. Themethod 200 further includes inserting the closure 18 into the open end14 of the tube 12 (206). The method 200 further includes drawing avacuum inside the tube 12 to a predetermined level (208) usingart-disclosed methods. The amount of vacuum to be drawn is dependentupon the nature and volume of the cells desired to be collected andpreserved. For example, for whole blood collection, the vacuum should bedrawn to a level that allows the pressure of the whole blood to cause itto flow into and fill the tube 12 to the desired level. The method 200may optionally include providing the components 32 conventionally usedto collect and analyze the cells 20. The components 32 are the same asfor the device 100 of the claimed subject matter as described above.

The method may also optionally include collecting the cells 20 usingart-disclosed methods (e.g., venipuncture, use of a lancet, etc.). Itmay optionally include screening the cells 20 using art-disclosedinstruments such as flow cytometers (eg., FACScan, FACSCalibur by BD andEPICS by Beckman Coulter), other hematology instruments (e.g., H3 byBayer Corporation, the Beckman Coulter STKS or Gen-S Systems, the AbbottCell-Dyn 4000 Hematology System, Bayer ADVIA 120 System, the SysmexXE2100 System, and the like. The screening of the cells may be for anypurpose including, without limitation, for HIV, HPV, hepatitis,leukemia, cancer, and the like; other art-disclosed screening such asimmunoassay, AIDS panel, and the like; and screening by methodsdisclosed in commonly U.S. Pat. No. 4,788,139 (Ryan) titled “Plateletaggregation reagent, reagent container and method of determiningplatelet aggregation in EDTA-anticoagulated blood”, which is herebyincorporated by reference. Cells collected and preserved using theclaimed subject matter may undergo histological study in any knownconventional manner, such as through the use of paraffin sectioningequipment, staining, mounting on slides, or other common steps utilizedprior to microscopic or other examination. The claimed subject matterthus provides a safe, convenient, and effective solution to collect andpreserve cells for analysis.

It should be noted that the method and device of the claimed subjectmatter may be used by those skilled in the art to preserve antigenicsites on or within cells (or components thereof) derived from any sourceincluding normal blood, bone marrow, lymph, or solid tissues, or may bederived from abnormal tissues such as leukemias or solid tissue cancers.The claimed subject matter may also be utilized with any cellularcomponent or biological material that has at least one antigenic site.

It should be noted that in preferred embodiments of the claimed subjectmatter cell clumping is prevented, light scattering properties arepreserved, antigenic sites are preserved, and nucleic acids may beanalyzed.

The foregoing detailed description has discussed only a few of the manyforms that the claimed subject matter can take. For this reason, thisdetailed description is intended only by way of illustration. It is onlythe following claims, including all equivalents that are intended todefine the scope of the claimed subject matter.

1. A method of preserving biological specimens for disease diagnosiscomprising: providing a tube having an open end and a closed end;preloading compounds including: a fixative agent into said tube, saidfixative agent capable of stabilizing abnormal cells such as cancercells; placing a biological specimen containing cancer cells into thetube; wherein said compounds prevent significant in vitro cell clumpingand dilution of said cells at room temperature. 2-27. (canceled)
 28. Themethod of claim 1, wherein the fixative agent is a formaldehyde donorcapable of releasing free formaldehyde.
 29. The method of claim 1,wherein the fixative agent is imidazolidinyl urea.
 30. The method ofclaim 1, wherein the preloaded compounds include polyethylene glycol.31. The method of claim 1, wherein the preloaded compounds include ananti-coagulating agent.
 32. The method of claim 31, wherein theanti-coagulating agent is a chelating agent.
 33. The method of claim 32,wherein the anti-coagulating agent is ethylenediamine tetraacetic acid(EDTA).
 34. The method of claim 31, wherein the fixative agent andanti-coagulating agent are combined prior to contact with the biologicalspecimen.
 35. The method of claim 34, wherein the anti-coagulating agentis a chelating agent.
 36. The method of claim 35, wherein theanti-coagulating agent is ethylenediamine tetraacetic acid (EDTA). 37.The method of claim 31, wherein the anti-coagulating agents and fixativeagents are present in volumes of about 0.5% to about 2% of the totalvolume of the biological specimen and compounds combined.
 38. A methodfor preserving blood samples suspected to contain cancer cellscomprising: a. obtaining a biological specimen that contains cells; b.contacting the biological specimen with a fixative agent capable ofstabilizing cancer cells; c. mixing the fixative agent and biologicalspecimen where the mixing prevents in vitro formation of cell clumps andin vitro cell dilution during storage at room temperature.
 39. Themethod of claim 38, wherein the fixative agent is a formaldehyde donorcapable of releasing free formaldehyde.
 40. The method of claim 38,wherein the fixative agent is imidazolidinyl urea.
 41. The method ofclaim 38, wherein the preloaded compounds include polyethylene glycol.42. The method of claim 41, wherein the preloaded compounds include ananti-coagulating agent.
 43. The method of claim 42, wherein theanti-coagulating agent is a chelating agent.
 44. The method of claim 43,wherein the anti-coagulating agent is ethylenediamine tetraacetic acid(EDTA).
 45. The method of claim 42, wherein the fixative agent andanti-coagulating agent are combined prior to contact with the biologicalspecimen.
 46. The method of claim 45, wherein the anti-coagulating agentis a chelating agent.
 47. The method of claim 46, wherein theanti-coagulating agent is ethylenediamine tetraacetic acid (EDTA). 48.The method of claim 42, wherein the anti-coagulating agents and fixativeagents are present in volumes of about 0.5% to about 2% of the totalvolume of the biological specimen and compounds combined.